Prodrugs of protein tyrosine kinase inhibitors

ABSTRACT

A compound which comprises a prodrug of a protein tyrosine kinase inhibitor (PTKi) linked to a protecting group which is capable of being cleaved from the compound to release the PTKi, said PTKi prodrugs including tyrphostins, of formula (I), ##STR1## where X represents C, N or a group N→O, n is 1 to 3; each group R 1 , which may be the same or different is H, OH, mercapto, carboxy, formyl, C 1-4  alkyl, C 2-4  alkenyl, C 1-4  alkoxy, C 1-4  alkylthio, carboxyC 1-4  alkyl, carboxyC 2-4  alkenyl, C 1-4  alkylsulphoxy, halo, nitro, amino, C 1-4  alkylamino, or C 1-4  dialkylamino, or when n is 2 or 3 two R 1  groups may together form a methylenedioxy or ethylenedioxy group; R 2  is H, OH, C 1-4  alkyl or together with position 2 of the ring to which the group(s) R 1  is (are) attached forms a 5 or 6 membered aliphatic or heterocyclic ring, optionally containing a ketone group; and R 3  is cyano, carboxy, carbamoyl, thiocarbamoyl, C(O)HNCH 2  CN, C(NH 2 )═C(CN 2 ), an alpha keto C(O)R 4  where R 4  is 3,4-dihydroxyphenyl or 2-thiophene or an alpha amido C(O)NHR 5  where R 5  is benzyl, phenyl, or 2,4-dimethoxyphenyl; provided that at least one of R 1  and R 2  is mercapto, hydroxy or amino.

This application is a filing under 35 U.S.C. 371 of PCT/GB94/01532 filed15 Jul. 1994.

FIELD OF THE INVENTION

The present invention relates to prodrugs and their use in the treatmentof tumours.

TECHNOLOGY REVIEW

The use of prodrugs represents a clinically very valuable concept incancer therapy since, particularly where the prodrug is to be convertedto an anti-tumour agent under the influence of an enzyme that islinkable to a monoclonal antibody that will bind to a tumour associatedantigen, the combination of such a prodrug with such an enzymemonoclonal/antibody conjugate represents a very powerful clinical agent.This approach to cancer therapy, often referred to as "antibody directedenzyme/prodrug therapy" (ADEPT) is disclosed in WO88/07378.

More recently, a similar approach ("VDEPT") has been proposed where inplace of an antibody/enzyme conjugate, tumour cells are targeted with aviral vector carrying a gene encoding an enzyme capable of activating aprodrug. The gene may be transcriptionally regulated by tumour specificpromoter or enhancer sequences. The viral vector enters tumour cells andexpresses the enzyme, in order that a prodrug is converted to an activedrug only in the vicinity of the tumour cells (Huber et al, Proc. Natl.Acad. Sci. USA (1991) 18, 8039). Alternatively, non-viral methods forthe delivery of genes have been used. Such methods include calciumphosphate co-precipitation, microinjection, liposomes, direct DNAuptake, and receptor-mediated DNA transfer. These are reviewed in Morgan& French Anderson, Annu. Rev. Biochem., 1993, 62;191. The term "GDEPT"(gene-directed enzyme prodrug therapy) is used to include both vital andnon-viral delivery systems.

Although the GDEPT and ADEPT systems enhance the concentrations ofanti-tumour agents which may be delivered to the site of a tumour, thereis still a need to enhance the specificity of drug delivery. In bothsystems, active drug can be released into the environment of normalcells and cause damage. In the case of ADEPT, this can be caused byactivation of prodrug by conjugates which have failed to localise at thetumour site. In GDEPT, transformation of normal tissue may lead toresidual levels of expression of the enzyme away from the tumour oractive drug may be released from tumour cells. .Although ways toincrease the specificity of the ADEPT system is disclosed in WO89/10140,there remains a continuing need to improve the level of ADEPT and GDEPTspecificity.

SUMMARY OF THE INVENTION

The present invention addresses such problems by the use of a novelclass of prodrugs, which are prodrugs of protein tyrosine kinase (PTK)inhibitors. Some PTKs are known to be over-expressed by some types oftumours such as in breast and ovarian carcinomas, where the cErbB2 geneis over-expressed. Certain compounds have been found to be selective forPTKs and thus are relatively non toxic to cells which do notover-express PTKs.

The use of such compounds in ADEPT or GDEPT thus provides an increasedlevel of specificity for the treatment of tumour cells. Prodrugs basedupon PTK-inhibitors will be converted into PTK inhibitors primarily atthe site of a tumour, but at the same time release of PTK-inhibitors atother sites or from the tumour will not cause cytotoxicity comparable tothe release of non-specific cytotoxic drugs.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a compound which comprisesa prodrug of a protein tyrosine kinase inhibitor (PTKi) linked to atleast one protecting group said group being capable of being cleavedfrom said compound to release the protein tyrosine kinase inhibitor or aphysiologically acceptable derivative of said prodrug.

The prodrug is of the general formula:

    PTKi-PRT

where PTKi compound is a compound with PTK inhibitory activity and PRTis at least one protecting group capable of being cleaved from the PTKinhibitor by the action of an enzyme.

Suitable PTKs include tyrphostins. Tyrphostins are low molecular weight(e.g. less than 2,000) styryl containing inhibitors of protein tyrosinekinase which are capable of binding to the subsite of protein tyrosinekinase domains. Suitable tyrphostins include those described by Gazit etal (Gazit et al, J. Med. Chem. (1989) 32, 2344) and Gazit et al (J. Med.Chem. (1991) 43; 1896-1907) and especially tyrphostins of the generalformula (I) ##STR2## where X represents carbon, a nitrogen or a groupN→O, n is an integer from 1 to 3;

each group R¹, which may be the same or different is hydrogen, hydroxy,mercapto, carboxy, formyl, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ alkoxy, C₁₋₄alkylthio, carboxyC₁₋₄ alkyl, carboxyC₂₋₄ alkenyl, C₁₋₄ alkylsulphoxy,halo (ie. fluoro, chloro, bromo or iodo), nitro, amino, C₁₋₄ alkylamino,or C₁₋₄ dialkylamino, or when n is 2 or 3 two R¹ groups may togetherform a methylenedioxy or ethylenedioxy group;

R² is hydrogen, hydroxy, C₁₋₄ alkyl or together with position 2 of thering to which the group(s) R¹ is(are) attached forms a 5 or 6 memberedaliphatic or heterocylic ring, said 5 or 6 membered ring optionallycontaining a ketone group; and R³ is cyano, carboxy, carbamoyl,thiocarbamoyl, a group C(O)HNCH₂ CN, a group C(NH₂)═C(CN)₂, an alphaketo group C(O)R⁴ where R⁴ is 3,4-dihydroxyphenyl or 2-thiophenyl or analpha amido group C(O)NHR⁵ where R⁵ is benzyl, phenyl or2,4-dimethoxyphenyl;

provided that at least one of the groups R¹ and R² is mercapto, hydroxyor amino.

In a preferred embodiment, X is C; n is an integer from 1 to 3; eachgroup R¹, which may be the same or different is hydrogen, hydroxy,carboxy, formyl, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₁₋₄ alkoxy, carboxyC₁₋₄alkyl, carboxyC₂₋₄ alkenyl, halo (ie. fluoro, chloro, bromo or iodo),nitro, amino, C₁₋₄ alkylamino, or C₁₋₄ dialkylamino, or when n is 2 or 3two R¹ groups may together form a methylenedioxy or ethylenedioxy group;R² is hydrogen, hydroxy or C₁₋₄ alkyl; and R³ is cyano, carboxy,carbamoyl, thiocarbamoyl, a group C(O)HNCH₂ CN or a group C(NH₂)═C(CN)₂.

Most preferably, X represents carbon, n is an integer from 1 to 3; eachgroup R¹, which may be the same or different is hydrogen, hydroxy oramino; R² is hydrogen or hydroxy; and R³ is cyano, a group C(O)HNCH₂ CN,a group C(NH₂)═C(CN)₂, an alpha keto group C(O)R⁴ where R⁴ is3,4-dihydroxyphenyl, or an alpha amido group C(O)NHR⁵ where R⁵ isbenzyl; provided that at least one of the groups R¹, R², and R³ arehydroxy or amino.

Preferably, R¹ is hydroxy or amino.

When R² forms a 5 or 6 membered ring with R¹ preferred rings includeheterocyclic rings wherein the ring contain one nitrogen atom and 4 or 5carbon atoms. The total number of atoms includes the 2 carbon atoms ofthe ring to which the group (s) R¹ is (are) attached.

Suitable tyrphostins such as the above may be obtained by the methodsdisclosed in, or analogous to those of, Gazit et al 1989 and 1991, ibid,which are incorporated herein by reference.

Other PTK inhibitors include flavonoids, erbstatin, benzoquinoidansamycin antibiotics and various peptide and nucleotide analogues. Theexact nature of the PTKi will depend upon the particular target tumourfor which the PTKi is to be used, taking into acount the nature of theparticular PTK involved. This can be determined by those of skill in theart, for example by culture of a biopsy sample of the tumour in thepresence of a range of candidate PTKs. Suitable PTKs may be found inWorkman et al, Seminars in Cancer Biology, Vol 3 (1992), 369-381.

PTK inhibitors may be linked to any suitable protecting group which isremovable by an enzyme. Examples of such groups include those found inWO88/07378 or in WO93/08288. For example, WO93/08288 describes "selfimmolative" prodrugs which can be activated by the action of anitroreductase enzyme. These prodrugs are derivatives ofp-nitrobenzyloxycarbonyl compounds.

The exact structure of the protecting group will depend upon the natureof the ADEPT or GDEPT system with which a tyrphostin prodrug is to beused. It may be any suitable group which can be removed by an enzyme ormodified by the enzyme in such a manner that the group is unstable andundergoes "self immolation" to provide the active PTK.

The number of protecting groups attached to each PTKi will depend inpart upon the exact structure of the inhibitor compound. It will alsodepend upon the relative activity of the unprotected PTKi to the PTKiwhen different numbers of protecting groups are added, since ifadditional protecting groups will achieve a reduction in potency of theprodrug this will increase the ratio of activity of PTKi to PTKi-PRT.

Desirably, one or two protecting groups will be attached to each PTKimolecule to provide a compound of the invention, although more, e.g. 3,4 or 5 groups may be added where the PTKi is of a structure which willallow this number to be linked.

Accordingly, prodrugs according to the invention include compounds witha protecting group of the formula (II):

    PTK--(X--CO.O--CH.sub.2 --Ph--NO.sub.2).sub.m              (II)

where X is NH, O or S, m is an integer from 1 to 5 (e.g. 1, 2 or 3), Phis an optionally substituted phenylene ring and PTK is a group such thatPTK--(XH)_(m) is a PTKi containing m --XH groups. The nitro group may bein the 2-position although is desirably in the 4-position of the ringrelative to the Ph ring.

Within each compound of formula (II) where m from 2 to 5, each group Xand Ph may be the same or different. Preferably, they are the same.

PTK inhibitors of formula (II) include tyrphostins including those offormula (I) above in which at least one of the groups R¹ and R² is anhydroxy, mercapto or amino group.

Suitable substituents of the phenylene ring include 1 to 4 groups whichmay be the same or different which are selected from fluorine, chlorine,bromine, iodine, hydroxy, mercapto, amino, nitro, C₁₋₄ alkyl, C₁₋₄haloalkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, C₂₋₄ alkenyl, C₂₋₄ alkynyl, andC₂₋₄ haloalkenyl. Prefeably, the substituents are from 1 to 4 fluorinesin the 2, 3, 5 and 6 positions of the ring.

Preferred prodrugs according to the invention are those of formula(III):

    PTK--(O--CO.O--CH.sub.2 --Ph--NO.sub.2).sub.m              (III)

where m and Ph is as defined above and PTK is a group such thatPTK--(OH)_(m) is a PTKi compound containing m hydroxyl groups. Suchprodrugs include those tyrphostins of formula (I) above in which atleast one of the groups R¹, R² or R³ is a hydroxyl group. The nitrogroup may be in the 2-postition although is desirably in the 4-positionof the ring relative to the Ph ring.

Compounds of formulae (II) and (III) may be used as prodrugs in an ADEPTor GDEPT system in conjunction with a nitroreductase enzyme, includingthe E.coli nitroreductase described in WO93/08288. While the presentinvention is not dependent, for its definition, upon the exact mode ofaction of the nitroreductase on the compound of formula II or III, it isbelieved that the nitro group of the optionally substitutedp-nitrophenyl-benzyloxy-carbonyl residue is converted to thecorresponding amino or hydroxylamino group and that the resultingoptionally substituted p-aminobenzyloxy-carbonyl or optionallysubstituted p-hydroxyl-aminobenzyloxycarbonyl compound automaticallydegrades under the reaction conditions used for the enzymatic reductionto release the cytotoxic compound and form optionally substitutedp-aminobenzyl alcohol or optionally substituted p-hydroxylaminobenzylalcohol and carbon dioxide as by products in accordance with thefollowing reaction scheme: ##STR3##

The optionally substituted p-nitrobenzyloxycarbonyl compounds of theinvention are conveniently prepared by methods of chemical synthesisknown per se. For example, the amine or hydroxy PTKi compounds can bereacted with optionally substituted 4-nitrobenzyl chloroformate underanhydrous conditions in the presence of a hydrogen chloride acceptor,particularly an alkylamine such as triethylamine. This reaction can becarried out in a dry aprotic organic solvent such as THF or chloroformand the resulting compound of the invention of formula II or formula IIIpurified from the organic solvent by conventional methods such aschromatography or recrystallization. For use in ADEPT the prodrug shouldbe unable to or have limited ability to enter cells, whereas for GDEPTthe prodrug should enter cells. Accordingly, modifications may be madein the prodrug, eg in the benzene ring, to make the prodrug more, orless, lipophilic.

Similar prodrugs which can be activated by a carboxypeptidase enzymesuch as carboxypeptidase G2 (CPG2) can be made using benzyl haloformate(where halo is fluoro, chloro or bromo, preferably chloro) derivativesof the formula (IV):

    Q--CO.0.CH.sub.2 --Ph--Z--NH--glu                          (IV)

where Q is hydrogen or fluoro, chloro or bromo, Ph is as defined above,Z is --O.CO-- or --NH.CO-- and glu is the residue of glutamic acid, ie agroup:

    --CH(CO.sub.2 H)(CH.sub.2 CH.sub.2 CO.sub.2 H)

or a di--C₁₋₆ alkyl ester (e.g. an ethyl or t-butyl ester) thereof, inorder to provide prodrugs of the formula (V):

    PTK--(X--CO.0.CH.sub.2 --Ph--Z--NH--glu).sub.m             (V)

and of the formula (VI)

    PTK--(O--CO.0.CH.sub.2 --Ph--Z--NH--glu).sub.m             (VI)

where PTK is the residue of a PTKi compound such that PTK--(XH)_(m) andPTK--(OH)_(m) are as defined above, and where m, Ph, Z and glu are alsoas defined above. As mentioned above in connection with prodrugs offormula (II) and formula (III), for ADEPT the prodrug should havelimited ability to enter cells whereas for GDEPT the prodrug may bemodified if need be to make it more lipophilic in order that it doesenter cells. The gamma carboxylic group of the glutamic acid may bealtered to make compounds that are more lipophilic, e.g. with anaromatic or heterocyclic amide.

Within each compound of formula (V) where m from 2 to 5, each group Xand Ph may be the same or different. Preferably, they are the same.

In compounds of formulae (IV), (V) and (VI), the group --Z-- is in the4-position of the ring relative to the PTK containing substituent.

Compounds of the formula (V) and (VI) in which the PTK is a tyrphostin,especially a tyrphostin of formula (I) are preferred.

The benzyl chloroformate derivatives of the formula (IV) in which Z is--NH.CO-- may be made from 4-(chloromethyl)phenyl isocyanate by reactionof glutamic acid or a protected derivative thereof, eg in which bothcarboxy groups of the glutamic acid residue are protected with C₁₋₆alkyl such as ethyl or t-butyl groups. Suitably, the reaction is carriedout in a solvent such as CH₂ Cl₂ at about room temperature. Theresulting intermediate,4-chloromethyl!phenyl-ureidoglutamate-di-tert-butylester, is treated inaqueous ethanol under reflux to provide the corresponding4-hydroxymethyl compound and this is reacted with triphosgene ((CCl₃ O)₂CO) in an inert solvent, eg. THF, at room temperature to provide anoptionally protected compound of formula (IV). The compound whenprotected may be deprotected by treatment with trifluoroacetic acid orformic acid.

The benzyl chloroformate derivatives of the formula (IV) in which Z is--O.CO-- may be made starting from 4-hydroxybenzaldehyde. Briefly, thealdehyde is treated with 1,2-ethane dithiol in borane trifluoroetherateplus CH₂ Cl₂ at 25° C. for about 12 hours to form the 1,3 dithiolaneintermediate which is treated with triphosgene as above to form the 41,3 dithiolane! phenylchloroformate. This is coupled withditertbutyl-glutamate hydrochloride in dry THF in the presence oftriethylamine at room temperature for about 5 hours, to provide 4 1,3dithiolane! phenylcarbamate-glutamate-di-tbutyl. The dithiolane isdeprotected with mercuric perchloroate in methanol or THF andcholoroform at about 25° C. for about 5 minutes. The aldehyde isconverted to the corresponding benzylic alcohol by mild reduction withsodium borohydride or other mild reducing agents at room temperature inether and then converted to the corresponding chloroformate withtriphosgene as described above.

Compounds of the formula (V) and (VI) may be made from PTK inhibitorswhich contain an amino or hydroxy group by analogous procedures to themethods described above for the production of compounds of formulae (II)and (III).

PTK prodrugs of the formulae (V) or (VI) will be activated bycarboxypeptidases such as CPG2 by the action of the enzyme to remove theglutamic acid residue followed by "self immolation" of the remainingprodrug in a manner analogous to that described above in relation to thenitroreductases.

Prodrugs of the formula (V) where Z is --NH.CO-- may also be made usingnovel linkers of the formula (VII):

    HOH.sub.2 C--Ph--NH--CO--NH--glu                           (VII)

where Ph and glu are as defined above. The optionally substitutedphenylene group is substituted at the 4-position by the glu-containingmoiety relative to the hydroxymethyl group.

Thus in a further aspect, the invention provides novel linkers of theformula (VII). The linkers may also be linked to other pharmaceuticalcompounds containing a free hydroxy, amino or mercapto group to providenovel prodrugs and such prodrugs form an additional aspect of theinvention. The novel prodrugs may be prepared as pharmaceuticalcompositions and may be used in the treatment of patients using ADEPT orGDEPT as described herein.

The novel linkers of formula (VII) may be made from optionallysubstituted 4-nitrobenzyl alcohol, where the optional substituents areas defined for the group --Ph-- above. The hydroxyl group of the4-nitrobenzyl alcohol is protected, for example by reaction withtert-butyl-diphenyl-chloro-silane at room temperature in an organicsolvent, to provide an optionally substituted (4-nitro-benzyl)tert-butyl-di-phenyl-silyl ether. The 4-nitro group is then reduced toan amine group by catalytic hydrogenation or catalytic hydrogentransfer, for example with ammonium formate in the presence of acatalyst such as Pd/C in a protic solvent such as an alcohol, e.g.methanol or ethanol.

The amine group may then converted into an isocyanate group for exampleby reaction with phosgene, diphosgene or triphosgene in the presence ofa tertiary organic amine such as triethylamine and an aprotic organicsolvent with a boiling point higher than 50° C. such as toluene. Theisocyanate compound is then reacted with di-C₁₋₆ alkyl-glutamic acid orderivative thereof, eg di-C₁₋₆ alkyl-glutamate hydrochloride. This maybe done at room temperature in the presence of triethylamine in anaprotic organic solvent such as toluene, THF or dichloromethane.

Alternatively, the amine compound may be reacted directly in a one-potsynthesis with the di-C₁₋₆ alkyl-glutamic acid or derivative thereof inthe presence of triphosgene and triethylamine in an aprotic solvent suchas THF or dichloromethane.

In either case, the resulting compound is treated to remove thehydroxy-protecting group, for example by the use of tetra-butylammoniumfluoride in THF at room temperature.

The resulting compound of formula (VII) where glu is in the form of adi-C₁₋₆ alkyl ester may be deprotected to remove the ester groups forexample by the use of an acid such as formic or trifluoro acetic acid.Alternatively, it may be linked to a PTKi containing a group --OH, --NH₂or --SH by reaction with the PTKi or activated derivative thereof inaprotic solvents such as dichloromethane and/or THF in the presence of atertiary organic base such as triethylamine at room temperature, toprovide a compound of the formula (V). The di-C₁₋₆ alkyl ester groups ofthe compound, if present, may be removed as described above.

In order to link a PTKi with a group --XH to the novel linker of formula(VII) the group --XH may be converted to a reactive chloroformyl,chlorothioformyl or isocyanate derivative by the use of phosgene,diphosgene or triphosgene in the presence of a phase transfer catalystsuch as tetra-butyl ammonium hydrogen sulphate. The reaction may becarried out in the presence of a base such as NaOH in an organic solventsuch as toluene, THF or dichloromethane.

In a further aspect of the invention, prodrugs of the formula (V) inwhich Z is --O.CO-- may be made using novel linkers of the formula(VIII):

    HOH.sub.2 C--Ph--O--CO--NH--glu                            (VIII)

where Ph and glu are as defined above. The optionally substitutedphenylene group is substituted at the 4-position by the glu-containingmoiety relative to the hydroxymethyl group.

Thus in a further aspect, the invention provides novel linkers of theformula (VIII). The linkers may also be linked to other pharmaceuticalcompounds containing a free hydroxy, amino or mercapto group to providenovel prodrugs and such prodrugs form an additional aspect of theinvention. The novel prodrugs may be prepared as pharmaceuticalcompositions and may be used in the treatment of patients using ADEPT orGDEPT as described herein.

To produce a compound of formula (VIII), optionally substituted4-hydroxybenzaldehyde is protected as a 1,3-dithiane or dithiolanein inan aprotic solvent such as CH₂ Cl₂ in the presence of BF₃.Et₂ O, at roomtemperature by reaction with 1,3-propanedithiol or 1,2-ethanedithiol, togive the corresponding 4(1',3'-dithianyl) phenol or 4(1',3'-dithidanyl)phenol. This compound is coupled with di--C₁₋₆ alkyl-glutamylisocyanate, in an aprotic solvent such as toluene in the presence of atertiary organic amine such as triethylamine, to the corresponding 04(1',3'-dithianyl)-phenyl!N(di--C₁₋₆ alkyl-glutamyl)carbamate. Thedeprotection of the carbamate to the corresponding aldehyde, may becarried out with Hg(ClO₄)₂ or Tl(NO₃)₃ in THF or dichloromethane at roomtemperature. The reduction of the aldehyde yields the desiredO(4-benzyl-oxy)N(di--C₁₋₆ alkyl-glutamyl) carbamate. This may bedeprotected by treatment with an acid such as trifluoroacetic or formicacid to remove the alkyl ester protecting groups to provide a prodrug offormula (V).

The novel linkers of formula (VIII) may be attached to PTKi compounds orother pharmaceutical compounds containing a free hydroxy, amino ormercapto group in the same way as described above for the linkers offormula (VII). Thus the invention further provides a compound which is aprodrug of an active drug wherein the active drug has at least one freeamino, hydroxyl or mercapto group which is/are linked to one or more(e.g. from 1 to 5, e.g. 1, 2 or 3) linkers of the formulae (VII) or(VIII), each of which may be the same or different.

Other suitable PTKi prodrugs (including tyrphostins such as those offormula (I)) include those which are derivatized with a sugar or aβ-lactam derivative. For example, suitable linkers which may be attachedto PTK inhibitors of the type PTK--NH₂ or PTK--OH or PTK--SH describedabove are: ##STR4## where R is hydrogen or acetyl and Y is aryl such asphenyl, benzyl or tolulyl, and these may be made in an analogous mannerto the other prodrugs described above.

Any hydroxy, amino or mercapto group of a PTKi may be linked in themanner described above to provide a prodrug of the present invention. Ifdesired, more than one such group may be derivatized to make a prodrug.If however only a single hydroxy, mercapto or amino group is to bereacted to form a prodrug, any remaining groups of the PTK may beprotected with for example tbutyl or adamantyl groups (in the case ofhydroxyl) or butyloxycarbonyl groups in the case of amino. Suchprotecting groups may be attached using chemical processes known in theart. The groups of the PTKi to be reacted with the linker may bederivatized to the corresponding haloformate or isocyanate and thencoupled with the linkers such as those of formulae (VII) or (VIII).After the PTKi prodrug has been made, the protecting groups may beremoved by conventional means, eg by treatment with trifluoroaceticacid.

Physiologically acceptable derivatives of said prodrug include salts,amides, esters and salts of esters. Esters include carboxylic acidesters in which the non-carbonyl moiety of the ester grouping isselected from straight or branched chain C₁₋₆ alkyl, (methyl, n-propyl,,n-butyl or t-butyl); or C₃₋₆ cyclic alkyl (e.g. cyclohexyl). Saltsinclude physiologically acceptable base salts, eg derived from anappropriate base, such as alkali metal (e.g. sodium), alkaline earthmetal (e.g. magnesium) salts, ammonium and NR₄ (wherein R is C₁₋₄ alkyl)salts. Other salts include acid addition salts, including thehydrochloride and acetate salts. Amides include non-substituted andmono- and di-substituted derivatives.

The invention further provides pharmaceutical formulations. Suchformulations comprise a compound of the invention together with one ormore pharmaceutically acceptable carriers or diluents.

Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral or parenteral (e.g. intramuscular orintravenous) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

For example, formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the polypeptide to blood componentsor one or more organs.

Suitable liposomes include, for example, those comprising the positivelycharged lipid (N 1-(2,3-dioleyloxy)propyl!-N,N,N-triethylammonium(DOTMA), those comprising dioleoyl-phosphatidylethanolamine (DOPE), andthose comprising 3β N-(n',N'-dimethylaminoethane)-carbamoyl!cholesterol(DC-Chol).

The PTKi prodrugs of the present invention and the antibody/enzymeconjugate for ADEPT can be administered simultaneously but it is oftenfound preferable, in clinical practice, to administer the enzyme/agentconjugate before the prodrug, e.g. up to 72 hours or even 1 week before,in order to give the enzyme/agent conjugate an opportunity to localisein the region of the tumour target. By operating in this way, when theprodrug is administered, conversion of the prodrug to the cytotoxicagent tends to be confined to the regions where the enzyme/agentconjugate is localised, i.e. the region of the target tumour thepremature release of the PTKi agent is minimised.

In VDEPT the prodrug will usually be administered followingadministration of the modified virus encoding an enzyme. Typically, thevirus will be administered to the patient and then the uptake of thevirus by infected cells monitored, for example by recovery and analysisof a biopsy sample of targeted tissue. Similarly in GDEPT the prodrugwill usually be administered following the administration of a deliverysystem containing the gene encoding the enzyme.

In ADEPT the degree of localisation of the enzyme/agent conjugate (interms of the ratio of localized to freely circulating active conjugate)can be further enhanced using the clearance and/or inactivation systemsdescribed in WO89/10140. This involves, usually following administrationof the conjugate and before administration of the prodrug, theadministration of a component (a "second component") which is able tobind to the such part of the conjugate so as to inactivate the enzymeand/or accelerate the clearance of the conjugate from the blood. Such acomponent may include an antibody to the enzyme component of the systemwhich is capable of inactivating the enzyme.

The second component may be linked to a macromolecule such as dextran, aliposome, albumin, macroglobulin or a blood group O erythrocyte so thatthe second component is restrained from leaving the vascularcompartment. In addition or as an alternative, the second component mayinclude a sufficient number of covalently bound galactose residues, orresidues of other sugars such as lactose or mannose, so that it can bindthe conjugate in plasma but be removed together with the conjugate fromplasma by receptors for galactose or other sugars in the liver. Thesecond component should be administered and designed for use such thatit will not, to any appreciable extent, enter the extravascular space ofthe tumour where it could inactivate localised conjugate prior to andduring administration of the prodrug.

The exact dosage regime for both GDEPT and ADEPT will, of course, needto be determined by individual clinicians for individual patients andthis, in turn, will be controlled by the exact nature of the prodrug andthe cytotoxic agent to be released from the prodrug but some generalguidance can be given. Chemotherapy of this type will normally involveparenteral administration of both the prodrug and either theenzyme/agent conjugate or modified virus and administration by theintravenous route is frequently found to be the most practical. In ADEPTsystems, the dose of the prodrug and conjugate will ultimately be at thediscretion of the physician, who will take into account such factors asthe age, weight and condition of the patient. Suitable doses of prodrugand conjugate are given in Bagshawe et al. Antibody, Immunoconjugates,and Radiopharmaceuticals (1991), 4, 915-922. A suitable dose ofconjugate may be from 500 to 200,000 enzyme units/m² (e.g. 20,000 enzymeunits/m²) and a suitable dose of prodrug may be from 5 to 2000 mg/m²(e.g. 200 mg/m²).

In order to secure maximum concentration of the conjugate at the site ofdesired treatment, it is normally desirable to space apartadministration of the two components by at least 4 hours. The exactregime will be influenced by various factors including the nature of thetumour to be targeted and the nature of the prodrug, but usually therewill be an adequate concentration of the conjugate at the site ofdesired treatment within 48 hours.

In GDEPT systems, the amount of virus or other vector delivered will besuch as to provide a similar cellular concentration of enzyme as in theADEPT system mentioned above. This may be determined by clinical trialswhich involve administering a range of trial doses to a patient andmeasuring the degree of infection or transfection of a target cell ortumour. The amount of prodrug required will be similar to or greaterthan that for ADEPT systems.

The present invention also provides a system for use in the control ofneoplasia in a human or animal subject comprising an enzyme capable ofconverting a PTKi prodrug to an active PTKi, preferably conjugated witha targeting agent such as monoclonal antibody that will bind to atumour-associated antigen, in association with a prodrug as definedabove. When the enzyme is a nitroreductase, the system also preferablycomprises a suitable cofactor for the enzyme. Suitable cofactors includea riboside or ribotide of nicotinic acid or nicotinamide.

The present invention extends to a method of treating neoplasia in ahuman or animal subject requiring such treatment which comprisesadministering to the host an effective amount of a PTKi prodrug of theinvention and an enzyme, preferably conjugated with a targeting agentsuch as a monoclonal antibody that will bind to a tumour-associatedantigen.

The present invention also provides a system for use in the control ofneoplasia in a human or animal subject comprising a modified virus orother delivery system capable of selectively infecting tumour cells insaid subject, said virus carrying a DNA or RNA sequence encoding anenzyme, in association with a PTKi prodrug capable of being converted toa PTKi by the action of said enzyme.

The present invention extends to a method of treating neoplasia in ahuman or animal subject requiring such treatment which comprisesadministering to the host an effective amount of a PTKi prodrug of theinvention and a modified virus, said modified virus capable ofselectively infecting tumour cells in said subject, said virus carryinga DNA or RNA sequence encoding an enzyme capable of converting said PTKiprodrug to an active PTKi.

The present invention also extends to a method of treating neoplasia ina human or animal subject requiring such treatment which comprisesadministering to the host an effective amount of a PTKi prodrug of theinvention and a non viral vector system, said non-viral vector systemcapable of being selectively introduced into tumour cells in saidsubject, said vector system carrying a DNA or RNA sequence encoding anenzyme capable of converting said PTKi prodrug to an active PTKioperably linked to a promoter effective in expressing said enzyme insaid cells.

The various systems for use in the treatment of neoplasia by ADEPTdescribed above optionally include the "second component" foraccelerated clearance described above. Likewise, the methods oftreatment of neoplasia described above optionally include as part ofthat method the use of the second component, an effective amount ofwhich is administered after administration of the enzyme, in order toincrease the ratio of localised to freely circulating enzyme. Referencemay be made to WO89/10140 for further particular details of the secondcomponent, and such details can be incorporated for use in the presentinvention.

Modified viruses capable of selectively infecting tumour cells are knownin the art. By "selectively infecting" it is meant that the virus willprimarily infect tumour cells and that the proportion of non-tumourcells infected is such that the damage to non-tumour cells byadministration of the PTKi prodrug will be acceptably low, given thenature of the disease being treated. Ultimately, this will be determinedby the physician.

It will also be understood that the DNA or RNA sequence encoding anenzyme carried by the virus will be linked to suitable expressioncontrol signals such that expression of the enzyme will occur in thetargeted tumour cells.

The non-viral vector system will be capable of being selectivelyintroduced into tumour cells utilizing methods such as those mentionedabove, e.g. calcium phosphate co-precipitation, microinjection,liposomes, direct DNA uptake, and receptor-mediated DNA transfer (Morgan& French Anderson, Annu. Rev. Biochem., 1993,62;191).

Suitable monoclonal antibodies for use in the present invention includeantibodies to cerbB2, such as ICR12 (Bakir, M A et al, J. Nucl. Med(1992) 33;2154-2160), and antibodies to epidermal growth factorreceptor, such as ICR16 (Dean, C J et al, Int. J. Cancer Suppl.8,(1994), 103).

As used herein, the term "monoclonal antibody" will be understood bythose of skill in the art not simply to refer to antibodies produced bytraditional hybridoma techniques, but also to cover antibodies andvariants thereof produced by recombinant means. These include, forexample, humanised antibodies such as those with a constant region froma human antibody grafted onto a non-human antibody variable region (seefor example EP-A-O 120 694), chimeric antibodies such as those withnon-human complementarity determining regions (CDRs) grafted into ahuman variable region framework (see for example EP-A-0 239 400) andsingle chain antibodies. Fragments of such monoclonal antibodies whichretain their target binding activity are also included by the generalterm "monoclonal antibody". This includes Fv, Fab' and F(ab')₂fragments. It also includes recombinant or synthetic proteins based uponthe CDRs of such antibodies, e.g. abzymes (a polypeptide with bothantibody-like binding acitivity and enzyme activity) and diabodies.

Prodrugs of the present invention may also be used as reagents in invitro systems to test the activity of candidate enzymes or antibodieswhich may be incorporated into ADEPT or GDEPT systems.

For example, a tumour cell line carrying a marker to which an antibodyis directed may be grown in vitro, and then an antibody-enzyme conjugateadded to the culture. The enzyme will be one which is, or suspected tobe, capable of converting a prodrug of the invention into an activedrug. The prodrug is then added to the culture and the amount of cellkilling or inhibition of cell growth is measured (for example by using avital stain to record the number of viable cells or by replating asample of the culture to count the number of viable cells).

EXAMPLES

The following examples illustrate the invention. The reaction schemeswhich follow further illustrate these examples.

All starting materials, reagents and anhydrous solvents (THF under N₂)were purchased from Aldrich, unless otherwise stated. The di-tert-butylglutamate is commercially available from Sigma. Kiselgel 60(0.043-0.060) was used in gravity columns (Art 9385 and 15111, Merck).TLC was performed on precoated sheets of Kiselgel 60 F₂₅₄ (Art 5735,Merck). Electron Impact spectra were determined with a VG 7070H massspectrometer and a VG 2235 data system using the direct-insertionmethod, an ionizing energy of 70 eV, trap current of 100 mA and anion-source temperature at 180°-200° C. FAB mass spectra were determinedusing xenon gas. High resolution accurate mass spectra were determinedon the same systems. Reported spectra are by FAB unless otherwisestated. NMR spectra were determined in Me₂ SO--d₆ on a Brucker AC250spectrometer (250 MHz) at 30° C. (303 K) unless otherwise stated. I.R.spectra (film) were recorded on a Perkin Elmer 1720X FT-I.R.spectrometer.

EXAMPLE 1

Summary

A tyrphostin prodrug, N¹ (4-hydroxybenzyl)N³ (di-tert-butyl-glutamyl)urea, 8 (See Scheme 1) clearable by the enzyme CPG2 was made. Thiscompound is designed to be coupled to the hydroxy, mercapto or aminofunctional groups of tyrphostin compounds. The intermediate N¹(4-hydroxybenzyl)N³ (di-tert-butyl-glutamyl) urea 8, was synthesised forcoupling to tyrphostin drugs according to the Scheme 1. The startingmaterial, 4-nitrobenzylic alcohol, 1, was protected astert-butyl-di-phenyl-silyl ether, 2, by reacting withtert-butyl-diphenyl-chlorosilane and imidazole in DMF (or THF) at roomtemperature. The protected nitro derivative, 2, was reduced by hydrogentransfer with ammonium formate (Pd/C 10% in EtOH). The amine, 3, thusformed was reacted with triphosgene in toluene at 70° C., to form thecorresponding isocyanate 4. The protected linker, 7, was obtained bycoupling the isocyanate 4 with di-tert-butyl-glutamate in THF in thepresence of NEt₃ at room temperature. An alternative route to 7 was bythe direct coupling of amine 3 with the di-tert-butyl-glutamylisocyanate 6, under the same conditions as described above, where thedi-tert-butyl-glutamyl isocyanate 6 was obtained from thedi-tert-butyl-glutamate by treatment with triphosgene and NEt₃ intoluene at -78° C. Using this route, the compound 7 was obtained in goodyield from the amine 3 and di-tert-butyl-glutamate in a one-potsynthesis.

The compound 7 was deprotected by Bu₄ NF in THF at room temperature andthe di-tert-butyl ester of the linker, 8, purified by columnchromatography. The ester 8 was reacted with4-chloroformyl-benzilydene-malononitrile, 10, resulting in thedi-tert-butyl ester linker of the tyrphostin 11. A phase transfercatalysis system was utilised since the phosgenation of the4-hydroxy-benzilydene-malononitrile 9, which would be the usualprocedure of choice, resulted only in the corresponding carbonate. Thephase transfer method used tetra-butylammonium hydrogen sulphate ascatalyst and led to a high yield of the desired compound. The finaldeprotection to compound 12 was carried out using formic acid at 4° C.

Experimental

(4-nitro-benzyl) tert-butyl-di-phenl-silyl ether (2)

To a stirred solution of 4-nitrobenzyl alcohol, 1, (1.00 g, 6.50 mmol),and imidazole (0.97 g, 14.1 mmol) in DMF (10.0 mL), was addedtert-butyl-diphenyl-chlorosilane (1.98 g, 7.20 mmol) over 10 min underN₂ at room temperature. The reaction mixture was stirred for anadditional 5 h, diluted with Et₂ O (75 mL), washed with H₂ O (5×15 mL),dried (MgSO₄) and evaporated to dryness under vacuum. An oil wasobtained which crystallised on standing and was recrystallised to asolid from EtOH (70%); yield: 2.36 g (93.%). v_(max) /cm⁻¹ (film): 2931,2857 (CH₂, asym., sym. ), 1521, 1345 (NO₂); ¹ H--NMR, d_(H) : 1.06(9H,s, t-Bu.), 4.92 (2H, s, CH₂), 7.42-7.46 (5H, m, Ph), 7.63-7.65 (7H, m,Ph+H_(arom2+6)), 8.23 (2H, d, J=8.23, H_(arom3+5)); MS, (EI), (391.54);m/z: 334 (M--t-Bu, 100), 288 (M--t-Bu--NO₂, 10), 256 (M--t-Bu--Ph, 20),199 (Ph₂ SiOH⁺, 100); C₂₃ H₂₅ NO₃ Si.

(4-amino-benzyl) tert-butyl-di-phenyl-silyl ether (3)

To a stirred solution of 2 (5.00 g, 12.77 mmol) in ethanol (100 mL) wasadded Pd/C (10%, 1.50 g) and ammonium formate (4.60 g) at roomtemperature. After 1.5 h the catalyst was removed by filtration, thefiltrate concentrated to dryness under vacuum and the residuepartitioned between EtOAc:H₂ O. The organic layer was dried (MgSO₄) andconcentrated under vacuum to give 3 as an oil; yield: 4.24 g (92%);v_(max) /cm⁻¹ (film): 3433, 3378 (NH₂), 2931, 2857 (CH₂, asym., sym.); ¹H--NMR, d_(H) : 1.00 (9H, s, t-Bu) , 4.57 (2H, s, CH₂) , 4.98 (2H, sbroad, NH₂) , 6.52 (2H, d, J=8.25, H_(arom3+5)) , 6.96 (2H, d,H_(arom2+6)) , 7.42-7.46 (5H, m, Ph), 7.62-7.65 (5H, m, Ph); MS, (EI),(361.56); m/z: 361 (M⁺, 8), 304 (M--t-Bu, 100), 199 (Ph₂ SiOH⁺, 100);C₂₃ H₂₇ NOSi.

(4-isocyanato-benzyl)tert-butyl-di-phenyl-silyl ether (4)

To a stirred solution of 3 (0.63 g, 1.70 mmol) and triethylamine (0.16g, 0.60 mmol) in toluene (10 mL) at 70° C., was added triphosgene (0.18g, 1.7 mmol). After 5 h the reaction mixture was filtered and thefiltrate evaporated to an oil under vacuum; yield: 0.65 g (99%) whichwas used without further purification; v_(max) /cm⁻¹ (film): 2931, 2857(CH₂, asym., sym.), 2275 (NCO); ¹ H--NMR, d_(H) : 1.03 (9H, s, t-Bu),4.76 (2H, s, CH₂) , 7.23 (2H, d, J=8.38, H_(arom3+5)) , 7.35 (2H, d,H_(arom2+6)) , 7.37-7.48 (5H, m, Ph) , 7.62-7.71 (5H, m, Ph); MS, (EI),(387.55); m/z: 330 (M--t-Bu, 52), 286 (M--t-Bu, M--t-Bu--NCO, 48), 199(Ph₂ SiOH⁺, 100); C₂₄ H₂₅ NO₂ Si.

N¹ (4 -tert-butyl-di-phenyl-silyl-O-benzyl) N³ (di-t-butyl-glutamyl)urea (7)

Method A: To a solution of di-tert-butyl-glutamate hydrochloride (0.46g, 1.55 mmol) in THF (7 mL) was added triethylamine (0.31 g 3.10 mmol).The isocyanate, 4, (0.60 g, 1.55 mmol) in dry THF (3 mL) was added tothe glutamate ester at room temperature. After 2 h the reaction mixturewas filtered and evaporated to dryness under vacuum. The product waspurified by column chromatography (EtOAc : cyclohexane 2:1) resulting inthe oil, 7; yield 0.53 g (53%). v_(max) /cm⁻¹ (film): 3359 (NH), 2932,2857 (CH₂, asym., sym.), 1729 (C═O, ester), 1670 (C═O, urea), 1154(C--O, str.); ¹ H--NMR, d_(H) : 1.03 (9H, s, t-Bu) , 1,40 (9H, s,t-Bu-glu) , 1.43 (9H, s, t-Bu-glu), 1.68-2.00 (2H, 2 m, CH(NH)CH₂) ,2.18-2.32 (2H, 2 m, CH₂ CO₂ --t-Bu), 4.08-4.12 (1H, m, CH(NH)CH₂), 4.68(2H, s, CH₂), 6.38 (1H, d, J=8.12, NH--glu) , 7.19 (2H, d, J=8.41,H_(arom3+5)), 7.32-7.47 (7H, m, Ph+H_(arom2+6)), 7.62-7.70 (5H, m, Ph),8.54 (1H, s, NH--Ph); MS, (EI), (646.90); m/z: 540 (M--t-Bu+1, 2), 534(M--2t-Bu+2, 5), 478 (M--3t-Bu+3, 100), 199 (Ph₂ SiOH⁺, 100); C₃₇ H₅₀ N₂O₆ Si.

Method B: (one pot synthesis of compound 7) To a solution ofdi-tert-butyl-glutamate hydrochloride (4.14 g, 14.0 mmol) andtriphosgene (1.39 g, 4.67 mmol) in toluene at -78° C., triethylamine(2.83 g, 28.0 mmol) in toluene (10 mL) was added dropwise over 30 min.The reaction was allowed to warm to room temperature. After 50 min, asolution containing (4-amino-benzyl)tert-butyl-diphenyl-silyl ether, 3(5.00 g, 13.8 mmol) and triethylamine (1.95 mL, 14.0 mmol) was addedover 5-10 min. After 20 h, the reaction mixture was filtered, washedsequentially with: H₂ O (200 mL), aq HCl (1%, 200 mL), aq Na₂ CO₃ (1%,200 mL), H₂ O (2×200 mL) dried (Mg₂ SO₄) and evaporated to an oil undervacuum; yield: 9.90 g. This product was deprotected without furtherpurification.

N¹ (4-hydroxyhenzyl)N³ (di-tert-butyl-glutamyl) urea (8)

From Method A- To a solution of 7, (0.53 g, 0.80 mmol) in THF (10 mL)was added tetra-butylammonium fluoride (2.5 mL, 2.5 mmol of 1M solution)in THF at room temperature. After 3 h, the reaction mixture wasevaporated to dryness under vacuum. The product was dissolved in EtOAc(20 mL) , washed with H₂ O (2×10 mL), dried (MgSO₄) and evaporated to anoil; yield: 0.40 g.

The deprotected compound, 8 (0.38 g), was purified by columnchromatography (EtOAc: cyclohexane 3:1) resulting in an oil whichcrystallised on standing; yield 0.093 g (29.%). v_(max) /cm⁻¹ (film):3370 (broad, NH+OH), 2967 (CH₃), 2930, 2857 (CH₂, asym., sym.), 1716(C═O, ester), 1678 (C═O, urea), 1153 (C--O, str.); ¹ H--NMR, d_(H) :1,40 (9H, s, t-Bu) , 1.42 (9H, s, t-Bu), 1.72-2.00 (2H, 2 m, CH(NH)CH₂), 2.20-2.31 (2H, 2 m, CH₂ CO₂ --t-Bu), 4.10-4.18 (1H, m, CH(NH)CH₂),4.39 (2H, d, J=5.36, CH₂), 4.99 (1H, t, CH₂ OH), 6.38 (1H, d, J=8.11,NH--glu), 7.16 (2H, d, J=8.35, H_(arom3+5)) , 7.31 (2H, d, H_(arom2+6)), 8.50 (1H, S, NH--Ph); MS, (EI), (408.94); m/z: 408 (M⁺, 10), 352(M--t-Bu+1, 4), 296 (M--2t-Bu+2, 14); C₂₁ H₃₂ N₂ O₆.

From Method B- The one pot procedure yielded 8, which was purified bycolumn chromatography; yield 2.57 g (46% over three steps) which wasrecrystallised from aq MeOH (60%).

(4-chloroformyl-benzylidene) malononitrile (10)

The Na salt of 4-chloroformyl-benzylidene malononitrile, 9 (0.34 g, 2.0mmol), was made in aq NaOH (10 mL, 0.10 g, 2.5 mmol ) To this solutionwas added the phase transfer catalyst, tetra-butyl ammonium hydrogensulphate (0.070 g, 0.2 mmol) in CH₂ Cl₂ (8 mL) with vigorous stirring.To this was added a solution of phosgene (20%, 0.40 g, 4.0 mmol) intoluene at room temperature. After 30 min the organic layer wasseparated, washed with H₂ O, dried (MgSO₄) and evaporated to a solidunder vacuum; yield: 0.39 g (84%). v_(max) /cm⁻¹ (film): 2230 (CN), 1784(C═O, chloroformate), 1199, 1166 (C--O, str.); ¹ H--NMR, (CDCl₃), d_(H): 7.50 (2H, d, J=8.81, H_(arom3+5)), 7,79 (1H, s, H_(vinyl)) , 8.02 (2H,d, H_(arom2+6)); MS, (EI) , (232.63); m/z: 232 (M⁺, 100); C₁₁ H₅ N₂ O₂Cl.

N¹ (4-benzylidene-malononitrile-oxy-carbonyl)-4-oxy benzyl)!N³(di-tert-butyl-glutamyl) urea (11)

To a solution of 10 (1.5 mmol) in CH₂ Cl₂ (10 mL) was added N¹(4-hydroxybenzyl)N³ (di-t-butyl-glutamyl) urea, 8 (0.41 g, 1.0 mmol) indry TMF (12.5 mL) and triethylamine (0.25 mL, 1.65 mmol) at roomtemperature under N₂. After 22 h, the reaction mixture was evaporated toa volume of 5 mL, dissolved in EtOAc (20 mL), washed sequentially withH₂ O (2×20 mL), aq NaOH (1%, 20 mL), H₂ O (2×20 mL), dried (MgSO₄) andevaporated under vacuum to an oil which was purified by columnchromatography (EtOAc: cyclohexane 3:1) resulting in a solid; yield 0.28g (65%) (0.12 g of the starting material 8 was recovered). v_(max) /cm⁻¹(film): 3369 (NH), 2924, 2857 (CH₂, asym., sym.), 1765 (C═O, carbonate),1728 (C═O, ester), 1658 (C═O, urea), 1216, 1153 (C--O, str.); ¹ H--NMR,d_(H) : 1,40 (9H, s, t-Bu) , 1.43 (9H, s, t-Bu) , 1.80-2.00 (2H, 2 m,CH(NH) CH₂), 2.22-2.35 (2H, 2 m, CH₂ CO₂ --t-Bu), 4.10-4.20 (1H, m,CH(NH) CH₂), 5.21 (2H, s, CH₂), 6.46 (1H, d, J=8.10, NH--glu), 7.33 (2H,d, J=8.48, H_(arom3+5) -cmpd 8), 7.42 (2H, d, H_(arom2+6) -cmpd 8), 7.53(2H, d, J=8.67, H_(arom3+5) -cmpd 9), 8.02 (2H, d, H_(arom2+6) -cmpd 9),8.54 (1H, s, NH--Ph), 8.68 (1 h, s, H_(vinyl)); MS, (604.59); m/z: 391(M--169--CO₂, 2), 279 (M--169--CO₂ -2t-Bu+2, 30) (169=cmpd 9-1); C₃₂ H₃₆N₄ O₈.

N¹ (4-benzylidene-malononitrile-oxy-carbonyl)-4-oxy benzyl)!N³ glutamylurea (12)

Compound, 11 (0.05 g,0.08 mmol) was dissolved in formic acid (95%, 6.0mL), at 4° C. under N₂. After 22 h the solvent was evaporated undervacuum (pump) to give a solid; yield: 0.037 g (91%). v_(max) /cm⁻¹(film): 3370 (v. broad, NH+OH), 2930, 2857 (CH₂, asym., sym. ) , 1719(C═O, ester) , 1681 (C═O, urea), 1221, 1176 (C--O, str.); ¹ H--NMR,d_(H) : 1.80-2.10 (2H, 2 m, CH(NH)CH₂) , 2.20-2.35 (2H, 2 m, CH₂ CO₂ H), 4.20-4.30 (1H, m, CH(NH)CH₂), 5.07 (2H, s, CH₂), 6.47 (1H, d, J=7.86,NH--glu), 6.80 (2H, d, J=8.76, H_(arom3+6) -cmpd 9), 7.25 (2H, d,J=8.39, H_(arom3+5) -cmpd 8) , 7.39 (2H, d, H_(arom2+6) -cmpd 8) , 7.89(2H, d, H_(arom2+6) -cmpd 9), 8.29 (1H, s, NH--Ph), 8.69 (1 h, s,H_(vinyl)); MS, (492.44); m/z: 323 (M--169, 18), 277 (M--169--CO₂, 18)(169=cmpd 9- 1); C₂₄ H₂₀ N₄ O₈.

EXAMPLE 2

Summary

Two tyrphostin prodrugs were designed which could be activated by theenzyme nitroreductase. These are4(4-nitro-phenyl-oxy-carbonyl)oxy-benzylidene-malononitrile, 15a, and3,4-di(4-nitro-phenyl-oxy-carbonyl)oxy-benzylidene-malononitrile, 15b,(See Scheme 2). For these syntheses,4-hydroxy-benzilydene-malononitrile, 13a, and3,4-dihydroxy-benzilydene-malononitrile, 13b were coupled with4-nitrobenzyl-chloroformate 14, leading to the desired prodrugs, 15a and15b respectively.

Experimental

4(4-nitro-phenyl-oxy-carbonyl)oxy-benzylidene-malononitrile (15a)

To a solution of 4-hydroxy-benzylidene-malononitrile, 13a (0.50 g, 2.93mmol), in dry THF (10 mL), was added 4-nitro-benzyl chloroformate, 14(0.63 g, 2.92 mmol) and triethylamine (0.30 g, 3.0 mmol) at an initialtemperature of 4° C. After 2.5 h, the reaction mixture was filtered andthe filtrate evaporated to dryness under vacuum. The residue thusobtained was partitioned against EtOAc: H₂ O (1:1, 25 mL), the organiclayer washed sequentially with aq NaOH (2%, 25 mL), aq HCl (2%, 25 mL)and H₂ O (2×25 mL), dried (MgSO₄) and evaporated to a solid undervacuum; yield: 0.55 g (54.%), which was recrystallised from EtOH.v_(max) /cm⁻¹ (film): 2230 (CN), 1767 (C═O, carbonate), 1522, 1349(NO₂), 1221 (C--O, str.); ¹ H--NMR, d_(H) : 5.46 (2H, s, CH₂) , 7.55(2H, d, J=8.77, H_(arom3+5)) , 7.73 (2H, d, J=8.70, H_(arom2+6) --PhNO₂), 8.03 (2H, d, H_(arom2+6)) , 8.27 (2H, d, H_(arom3+5) --PhNO₂), 8.55(1H, s, H_(vinyl)); MS, (EI), (349.30); m/z: 349 (M⁺, 3), 305 (M--CO₂,60); C₁₈ H₁₁ N₃ O₅.

3,4-di(4-nitro-phenyl-oxy-carbonyl)oxy-benzylidene-malono nitrile (15b)

A similar procedure was used for3,4-di-hydroxy-benzylidene-malononitrile, 13b (0.50 g, 2.70 mmol)resulting in a solid; yield: 0.86 g, (59.%) which was recrystallisedfrom EtOH. v_(max) /cm⁻¹ (film): 2232 (CN), 1776 (C═O, carbonate), 1523,1350 (NO₂), 1247 (C--O, str.); ¹ H--NMR, d_(H) : 5.44 (4s, CH₂), 7.66(4H, d, J=8.65, H_(arom2+6) -2PhNO₂), 7.79 (1H, d, J=8.40, H_(arom5)),7.99 (1H, q, H_(arom6)), 8.01 (1H, d, H_(arom2)), 8.18 (2H, d,H_(arom3+5) -PhNO₂ ') , 8.19 (2H, d, H_(arom3+5) -PhNO₂ ") , 8.56 (1H,s, H_(vinyl)).

EXAMPLE 3

Summary and Experimental

O(4-hydroxybenzyl)N(di-tert-butyl-glutamyl) carbamate, 20 (See Scheme3), another self-immolative linker, was prepared. Compound 16,4-hydroxy-benzaldehyde, was protected with 1,3-propane-dithiol in CH₂Cl₂ in the presence of BF₃.Et₂ O, at room temperature, to give the4(1',3'-dithianyl) phenol, 17, in good yield. Coupling of 17 withdi-tert-butyl-glutamyl isocyanate, 6, in toluene in the presence of Et₃N, led to the O4(1',3'-dithianyl)-phenyl!N(di-tert-butyl-glutamyl)carbam ate, 18. Thedeprotection of the carbamate, 18, to the corresponding aldehyde, 19,was carried out with Hg(ClO₄)₂ in THF at room temperature. The reductionof 19 yielded the desired O(4-benzyl-oxy)N(di-tert-butyl-glutamyl)carbamate, 20. This is coupled to the tyrphostin of 10 as describedabove in Example 1 and the ester protecting groups are removed. ##STR5##

What is claimed is:
 1. A compound of the formula (VII):

    HOH.sub.2 C--ph--NH--CO--NH--glu                           (VII)

where Ph is .an optionally substituted phenyl group and glu is theresidue of glutamic acid of the formula:

    --CH(CO.sub.2 H)(CH.sub.2 CH.sub.2 CO.sub.2 H)

or a di--C₁₋₆ alkyl ester thereof, and wherein the optionallysubstituted phenylene group is substituted at the 4-position by theglu-containing moiety relative to the hydroxymethyl group.
 2. A compoundof the formula (VIII):

    HOH.sub.2 C--Ph--O--CO--NH--glu                            (VIII)

where Ph is an optionally substituted phenyl group and glu is theresidue of glutamic acid of the formula:

    --CH(CO.sub.2 H)(CH.sub.2 CH.sub.2 CO.sub.2 H)

or a di--C₁₋₆ alkyl ester thereof, and wherein the optionallysubstituted phenylene group is substituted at the 4-position by theglu-containing moiety relative to the hydroxymethyl group.
 3. A compoundselected from the group consisting of:N¹ (4-hydroxybenzyl)N³(di-tert-butyl-glutamyl) urea; andO(4-benzyl-oxy)N(di-tert-butyl-glutamyl) carbamate.